Device for monitoring haptotaxis

ABSTRACT

The present invention discloses a device for monitoring haptotaxis including a housing defining a chamber. The chamber comprises: a first well region including at least one first well, the first well configured to receive a test agent therein and further including biomolecules immobilized therein; a second well region including at least one second well, the second well region configured to receive a sample comprising cells therein and further being horizontally offset with respect to the first well region in a test orientation of the device; and a channel region including at a least one channel connecting the first well region and the second well region with one another, the channel region further including biomolecules immobilized therein.

RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 09/709,776, filed on Nov. 8, 2000 now U.S. Pat. No.6,699,665 and claims the benefit of U.S. Provisional Application No.60/307,886, filed on Jul. 27, 2001; U.S. Provisional Application No.60/312,405, filed on Aug. 15, 2001; U.S. Provisional Application No.60/323,742, filed on Sept. 21, 2001; U.S. Provisional Application No.60/328,103; filed on Oct. 11, 2001; U.S. Provisional Application No.60/330,456, filed on Oct. 22, 2001; U.S. Provisional Application No.60/334,548, filed Dec. 3, 2001; and U.S Provisional Application No.60/363,355, filed on Mar. 12, 2002, all of which are herein incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to device for monitoringhaptotaxis.

BACKGROUND

Test devices, such as those used in chemotaxis, haptotaxis andchemoinvasion are well known. Such devices are disclosed for example inU.S. Pat. Nos. 6,329,164, 6,238,874, and 5,302,515.

Three processes involved in cell migration are chemotaxis, haptotaxis,and chemoinvasion. Chemotaxis is defined as the movement of cellsinduced by a concentration gradient of a soluble chemotactic stimulus.Haptotaxis is defined as the movement of cells in response to aconcentration gradient of a substrate-bound stimulus. Chemoinvasion isdefined as the movement of cells into and/or through a barrier or gelmatrix. The study of chemotaxis/haptotaxis and chemoinvasion and theeffects of external stimuli on such behavior are prevalent throughoutcontemporary biological research. Generally, this research involvesexposing a cell to external stimuli and studying the cell's reaction. Byplacing a living cell into various environments and exposing it todifferent external stimuli, both the internal workings of the cell andthe effects of the external stimuli on the cell can be measured,recorded, and better understood.

A cell's migration in response to a chemical stimulus is a particularlyimportant consideration for understanding various disease processes andaccordingly developing and evaluating therapeutic candidates for thesediseases. By documenting the cell migration of a cell or a group ofcells in response to a chemical stimulus, such as a therapeutic agent,the effectiveness of the chemical stimulus can be better understood.Typically, studies of disease It processes in various medical fields,such as oncology, immunology, angiogenesis, wound healing, andneurobiology involve analyzing the chemotactic and invasive propertiesof living cells. For example, in the field of oncology, cell migrationis an important consideration in understanding the process ofmetastasis. During metastasis, cancer cells of a typical solid tumormust loosen their adhesion to neighboring cells, escape from the tissueof origin, invade other tissues by degrading the tissues' extracellularmatrix until reaching a blood or lymphatic vessel, cross the basallamina and endothelial lining of the vessel to enter circulation, exitfrom circulation elsewhere in the body, and survive and proliferate inthe new environment in which they ultimately reside. Therefore, studyingthe cancer cells' migration may aid in understanding the process ofmetastasis and developing therapeutic agents that potentially inhibitthis process. In the inflammatory disease field, cell migration is alsoan important consideration in understanding the inflammatory response.During the inflammation response, leukocytes migrate to the damagedtissue area and assist in fighting the infection or healing the wound.The leukocytes migrate through the capillary adhering to the endothelialcells lining the capillary. The leukocytes then squeeze between theendothelial cells and use digestive enzymes to crawl across the basallamina. Therefore, studying the leukocytes migrating across theendothelial cells and invading the basal lamina may aid in understandingthe inflammation process and developing therapeutic agents that inhibitthis process in inflammatory diseases such as adult respiratory distresssydrome (ARDS), rheumotoid arthritis, and inflammatory skin diseases.Cell migration is also an important consideration in the field ofangiogenesis. When a capillary sprouts from an existing small vessel, anendothelial cell initially extends from the wall of the existing smallvessel generating a new capillary branch and pseudopodia guide thegrowth of the capillary sprout into the surrounding connective tissue.New growth of these capillaries enables cancerous growths to enlarge andspread and contributes, for example, to the blindness that can accompanydiabetes. Conversely, lack of capillary production can contribute totissue death in cardiac muscle after, for example, a heart attack.Therefore studying the migration of endothelial cells as new capillariesform from existing capillaries may aid in understanding angiogenesis andoptimizing drugs that block vessel growth or improve vessel function. Inaddition, studying cell migration can also provide insight into theprocesses of tissue regeneration, organ transplantation, autoimmunediseases, and many other degenerative diseases and conditions.

Cell migration assays are often used in conducting these types ofresearch. Commercially available devices for creating such assays aresometimes based on or employ a transwell system (a vessel partitioned bya thin porous membrane to form an upper compartment and a lowercompartment). To study cell chemotaxis, cells are placed in the uppercompartment and a migratory stimulus is placed in the lower compartment.After a sufficient incubation period, the cells are fixed, stained, andcounted to study the effects of the stimulus on cell chemotaxis acrossthe membrane.

To study chemoinvasion, a uniform layer of a MATRIGEL™ matrix is placedover the membrane to occlude the pores of the membrane. Cells are seededonto the MATRIGEL™ matrix in the upper compartment and a chemoattractantis placed in the lower compartment. Invasive cells attach to and invadethe matrix passing through the porous membrane. Non-invasive cells donot migrate through the occluded pores. After a sufficient incubationperiod, the cells may be fixed, stained, and counted to study theeffects of the stimulus on cell invasion across the membrane.

The use of transwells has several shortcomings. Assays employingtranswells require a labor-intensive protocol that is not readilyadaptable to high-throughput screening and processing. Because of theconfiguration of a transwell system, it is difficult to integrate withexisting robotic liquid handling systems and automatic image acquisitionsystems. Therefore, much of the screening and processing, such ascounting cells, is done manually which is a time-consuming, tedious, andexpensive process. Cell counting is also subjective and often involvesstatistical approximations. Specifically, due to the time and expenseassociated with examining an entire filter, only randomly selectedrepresentative areas may be counted. Moreover, even when these areas arecounted, a technician must exercise his or her judgment when accountingfor a cell that has only partially migrated through the filter.

Transwell-based assays have intrinsic limitations imposed by the thinmembranes utilized in transwell systems. The membrane is only 50-30microns (μm) thick, and a chemical concentration gradient that formsacross the membrane is transient and lasts for a short period. If a cellchemotaxis assay requires the chemotactic gradient to be generated overa long distance (>100-200 μm) and to be stable over at least two hours,currently available transwell assays cannot be satisfactorily performed.

Notwithstanding the above, perhaps the most significant disadvantage oftranswells is the lack of real-time observation of chemotaxis andchemoinvasion. In particular, the changes in cell morphology duringchemotaxis cannot be observed in real-time with the use of transwells.In transwells, when the cells are fixed to a slide, as required forobservation, they are killed. Consequently, once a cell is observed itcan no longer be reintroduced into the assay or studied at subsequentperiods of exposure to a test agent. Therefore, in order to study theprogress of a cell and the changes in a cell's morphology in response toa test agent, it is necessary to run concurrent samples that may beslated for observation at various time periods before and after theintroduction of the test agent. In light of the multiple samplesrequired for each test, in addition to the positive and negativecontrols required to obtain reliable data, a single chemotaxis assay canrequire dozens of filters, each of which needs to be individuallyexamined and counted-an onerous and time-consuming task.

More recently, devices for measuring chemotaxis and chemoinvasion havebecome available which employ a configuration in which two wells arehorizontally offset with respect to one another. This configuration of adevice was introduced by Sally Zigmond in 1977 and, hereafter referredto as the “Zigmond device,” consists of a 25 millimeters (mm) ×75 mmglass slide with two grooves 4 mm wide and 1 mm deep, separated by a 1mm bridge. One of the grooves is filled with an attractant and the othergroove is filled with a control solution, thus forming a concentrationgradient across the bridge. Cells are then added to the other groove.Two holes are provided at each end of the slide to accept pin clamps.The clamps hold a cover glass in place during incubation and observationof the cells. Because of the size and configuration of the Zigmondchamber, it does not allow integration with existing robotic liquidhandling systems and automatic image acquisition systems. Further, aswith transwell-based systems, the changes in cell morphology duringchemotaxis cannot be observed in real-time with the use of the Ziginondchamber as the cells are fixed to a slide for observation. In addition,the pin clamps must be assembled with an allen wrench and thus thedevice requires extra handling, positioning, and alignment beforeperforming the assay. Such handling and positioning of the cover glasson the glass slide, as well as the rigidity of the cover glass, canpotentially damage or interfere with any surface treatment on thebridge.

A chemotaxis device attempting to solve the problem of lack of real-timeobservation is the “Dunn chamber.” The Dunn chamber consists of aspecially constructed microslide with a central circular sink and aconcentric annular moat. In an assay using a Dunn chamber, cells migrateon a coverslip, which is placed inverted on the Dunn chamber, towards achemotactic stimulus. The cells are monitored over-night using aphase-contrast microscope fitted with a video camera connected to acomputer with an image-grabber board. In addition to the problems ofrigidity of the coverslip and the lack of integration into existingrobotic liquid handling systems, a major problem with the Dunn chamberassay is that only a very small number of cells are monitored (typicallyten). The average behavior of this very small sample may not be typicalof the population as a whole. A second major problem is that replicationis very restricted. Each control chamber and each treatment chamber mustbe viewed in separate microscopes, each one similarly equipped withcamera and computer.

Another chemotaxis device known in the art is disclosed in U.S. Pat. No.6,238,874 to Jarnagin et. al. (the '874 patent). The '874 patentdiscloses various embodiments of test devices that may be used tomonitor chemotaxis. However, disadvantageously, the devices in Jarnaginet al. can not be easily sealed or assembled or peeled and disassembled.Thus, it is difficult to maintain surfaces that are prepared chemicallyor biologically during assembly. The test devices of the '874 patent aretherefore more suited for one-time use. Also, disassembly and collectionof cells is difficult to do without damage to the cells or withoutdisturbing the cell positions.

The prior art has failed to provide a test device, such as a device formonitoring chemotaxis, haptotaxis, and/or chemoinvasion, which device iseasily assembled and dissembled. In addition, the prior art has failedto provide a test device for monitoring chemotaxis and/or chemoinvasion,which is not limited to measuring the effects on cell migration ofchemoattractants, chemorepellants and chemostimulants.

SUMMARY OF THE INVENTION

The present invention provides a device for monitoring haptotaxisincluding a housing comprising: a support member and a top member, thetop member mounted to the support member wherein the support member andthe top member are configured such that they together define a discretechamber. The discrete chamber includes a first well region including atleast one first well, the first well configured to receive a test agenttherein and further including biomolecules immobilized therein; a secondwell region including at least one second well, the second well regionconfigured to receive a sample comprising cells therein and furtherbeing horizontally offset with respect to the first well region in atest orientation of the device; and a channel region including at leastone channel connecting the first well region and the second well regionwith one another, the channel region further including biomoleculesimmobilized therein.

The present invention moreover provides a device for monitoringhaptotaxis including a housing defining a discrete chamber. The chamberhas an opening facing vertically upward in a test orientation of thedevice. The chamber further comprises: a first well region including atleast one first well, the at least one first well configured to receivea test agent therein and further including biomolecules immobilizedtherein; a second well region including at least one second well, thesecond well region further being horizontally offset with respect to thefirst well region in a test orientation of the device, the at least onesecond well configured to receive a sample comprising cells therein; anda channel region including at least one channel connecting the firstwell region and the second well region with one another, the at leastone channel further including biomolecules immobilized therein.

The present invention additionally provides a device for monitoringhaptotaxis. The device comprises support means and means mounted to thesupport means for defining a discrete chamber with the support means.The discrete chamber includes a first well region including at least onefirst well, the at least one first well configured to receive a testagent therein and further including biomolecules immobilized therein; asecond well region including at least one second well, the second wellregion further being horizontally offset with respect to the first wellregion in a test orientation of the device, the at least one second wellconfigured to receive a sample comprising cells; and a channel regionincluding at least one channel connecting the first well region and thesecond well region with one another, the at least one channel furtherincluding biomolecules immobilized therein.

The present invention also provides a device for monitoring haptotaxiscomprising a support member and a top member mounted to the supportmember by forming a substantially instantaneous seal with the supportmember. The support member and the top member are configured such thatthey together define a discrete chamber. The discrete chamber includes:a first well region including at least one first well, the at least onefirst well configured to receive a test agent therein and furtherincluding biomolecules immobilized therein; a second well regionincluding at least one second well, the second well region configured toreceive a sample comprising cells therein and further being horizontallyoffset with respect to the first well region in a test orientation ofthe device; and a channel region including at least one channelconnecting the first well region and the second well region with oneanother, the channel region further including biomolecules immobilizedtherein.

The present invention furthermore provides a device for monitoringhaptotaxis including a housing defining a chamber. The chambercomprises: a first well region including at least one first well, thefirst well configured to receive a test agent therein and furtherincluding biomolecules immobilized therein; a second well regionincluding at least one second well, the second well region configured toreceive a sample comprising cells therein and further being horizontallyoffset with respect to the first well region in a test orientation ofthe device; and a channel region including at a least one channelconnecting the first well region and the second well region with oneanother, the channel region further including biomolecules immobilizedtherein.

The present invention also provides a kit for monitoring haptotaxiscomprising: a device including a housing defining a chamber. The chambercomprises: a first well region including at least one first well, thefirst well configured to receive a test agent therein; a second wellregion including at least one second well, the second well regionconfigured to receive a sample comprising cells therein and furtherbeing horizontally offset with respect to the first well region in atest orientation of the device; and a channel region including at aleast one channel connecting the first well region and the second wellregion with one another. The kit further comprises a sample comprisingbiomolecules.

BRIEF DESCRIPTION OF THE DRAWINGS

The various features of the invention will best be appreciated bysimultaneous reference to the description that follows and theaccompanying drawings, in which:

FIG. 1A is a top, perspective view, in partial cross section, of aportion of an embodiment of test device according to the presentinvention;

FIG. 1B is a top, perspective view of an embodiment of a test device ofthe present invention;

FIG. 1C is a side-elevational view of a longitudinal cross section ofone of the chambers of the test device of FIG. 1B;

FIG. 2A is a schematic outline depicting a top plan view of analternative embodiment of a chamber defined in a test device of thepresent invention, where the channel region defines a single channel;

FIG. 2B is a schematic outline depicting a top plan view of theembodiment of the chambers defined in the embodiment of the test deviceaccording to FIG. 1B, where the channel region defines a single channel;

FIG. 2C is a figure similar to FIG. 2A, showing an alternativeembodiment of a chamber defined in a test device of the presentinvention, where the channel region defines a single channel;

FIG. 3A is a figure similar to FIG. 2A, showing an alternativeembodiment of a chamber defined in a test device of the presentinvention, where the channel region defines a plurality of channelshaving identical lengths;

FIG. 3B is a figure similar to FIG. 3A, showing a channel regiondefining a plurality of channels having lengths that increase from oneside of the chamber to another side of the chamber;

FIG. 3C is a figure similar to FIG. 3A, showing a channel regiondefining a plurality of channels having widths that increase from oneside of the chamber to another side of the chamber;

FIG. 4A is a figure similar to FIG. 1B showing an alternative embodimentof a test device according to the present invention;

FIG. 4B is an enlarged, schematic, top plan view of a channel of FIG. 4Ashowing cells on the sides of the channel;

FIGS. 5 and 6 are views similar to FIG. 2A, showing an alternativeembodiment of a chamber defined in a test device of the presentinvention, where the wells are trapezoidal in a top plan view thereof;

FIG. 7 is a view similar to FIG. 2A, showing an alternative embodimentof a chamber defined in a test device of the present invention, wherethe chamber is in the form of a FIG. 8 in a top plan view thereof;

FIG. 8 is a view similar to FIG. 2A, showing an alternative embodimentof a chamber defined in a test device of the present invention, whereone well is rectangular and the other well is circular in a top planview of the device;

FIG. 9 is a view similar to FIG. 2A, showing an alternative embodimentof a chamber defined in a test device of the present invention, wherethe first well region and the second well region each define a pluralityof wells, and where the channel region defines a plurality of channelsjoining respective wells of each well region;

FIG. 10 is a view similar to FIG. 2A, showing an alternative embodimentof a chamber defined in a test device of the present invention, wherethe channel region defines a plurality of channels joining respectivewells of each well region;

FIG. 11 is a view similar to FIG. 2A, showing an alternative embodimentof a chamber defined in a test device of the present invention, wherethe first well region has a plurality of wells and a respectivecapillary for each well, the channel region has a single channel, andthe second well region has a single well;

FIG. 12 is a side, cross-sectional view of an embodiment of a portion ofthe support member according to the present invention, the portion ofthe support member being shown along a longitudinal axis of a chamberaccording to the present invention;

FIG. 13 is an isometric view of a collective system according to oneembodiment of the present invention;

FIG. 14 is a view similar to FIG. 2A, showing an alternative embodimentof a chamber defined in a test device of the present invention, wherethe first well region includes a plurality of wells interconnected by anetwork of capillaries, where the channel region includes a singlechannel, and where the second well includes a single well;

FIG. 15 is a block diagram of an automated analysis system according toan embodiment of the present invention;

FIG. 16 is a flow diagram of a method according to an embodiment of thepresent invention;

FIG. 17 illustrates exemplary image data on which the method of FIG. 16may operate;

FIG. 18 illustrates a histogram that may be obtained from the image dataof FIG. 17;

FIG. 19 illustrates exemplary image data;

FIG. 20 illustrates exemplary dilated image data.

DETAILED DESCRIPTION OF THE EMBODIMENTS

As shown in FIG. 1A, according to one embodiment of the presentinvention, a test device 10, such as, for example, a cellularchemotaxis/haptotaxis and/or chemoinvasion device, includes a housing 10a comprising a support member 16 and a top member 11 mounted to thesupport member 16 by being placed in substantially fluid-tight,conformal contact with the support member 16. In the context of thepresent invention, “conformal contact” means substantially form-fitting,substantially fluid-tight contact. The support member 16 and the topmember 11 are configured such that they together define a discretechamber 12 as shown. Preferably test device 10 comprises a plurality ofdiscrete chambers, as shown by way of example in the embodiment of FIG.1B. The discrete chamber 12 includes a first well region 13 a includingat least one first well 13 and second well region 14 a including atleast one second well 14, the second well region further beinghorizontally offset with respect to the first well region in a testorientation of the device. The “test orientation” of the device is meantto refer to a spatial orientation of the device during testing. As shownin FIG. 1C, device 10 further includes a channel region 15 a includingat least one channel 15 connecting the first well region 13 a and thesecond well region 14 a with one another. In the embodiments of FIGS.1A-2C, each well region includes a single well, and the channel regionincludes a single channel. As seen in FIG. 1C, each well is defined by athrough-hole in top member 11, corresponding to well 13 and well 14respectively, and by an upper surface U of support member 16. Inparticular, the sides of each well 13 and 14 are defined by the walls ofthe through holes in the top member 11, and the bottoms of well 13 and14 are defined by the upper surface U of support member 16. It is notedthat in the context of the present invention, “top,” “bottom,” “upper”and “side” are defined relative to the test orientation of the device.As seen collectively in FIGS. 1A and 1C, a length L of channel region 15a is defined in a direction of the longitudinal axis of channel region15 a; a depth D of channel region 15 a is defined in a direction normalto upper surface U of support member 16; a width W is defined in adirection normal to the length L and depth D of channel region 15 a.According to one embodiment of the present invention, the chamber'sfirst well 13 is adapted to receive a test agent, a soluble testsubstance and/or a test agent comprising immobilized biomolecules, whichpotentially affects chemotaxis or haptotaxis. Biomolecules include, butnot limited to, DNA, RNA, proteins, peptides, carbohydrates, cells,chemicals, biochemicals, and small molecules. The chamber's second well14 is adapted to receive a biological sample of cells. Immobilizedbiomolecules are biomolecules that are attracted to support member 16with an attractive force stronger than the attractive forces that arepresent in the environment surrounding the support member, such assolvating and turbulent forces present in a fluid medium. Non-limitingexamples of the test agent include chemorepellants, chemotacticinhibitors, and chemoattractants, such as growth factors, cytokines,chemokines, nutrients, small molecules, and peptides. Alternatively, thechamber's first well 13 is adapted to receive a biological sample ofcells and the chamber's second well 14 is adapted to receive a testagent.

In one embodiment of the present invention, when a soluble testsubstance is used as the test agent, channel region 15 a preferablycontains a gel matrix. The gel matrix allows the formation of a solutionconcentration gradient from first well region 13 a towards second wellregion 14 a as the solute diffuses from an area of higher concentration(well region 13 a) through a semi-permeable matrix (the gel matrix) toan area of lower concentration (well region 14 a). If the soluble testsubstance comprises a chemoattractant, in order for the cells to migratethrough the matrix in the direction of the solution concentrationgradient towards well region 13 a, the cells must degrade this matrix byreleasing enzymes such as matrix metalloproteases. This cell chemotaxisand invasion may be subsequently observed, measured, and recorded.

In one embodiment of the present invention, utilizing immobilizedbiomolecules as the test agent, the biomolecules are preferablyimmobilized or bound on the portion of support member 16 underlyingchannel region 15 a and underlying through hole for well region 13 a.The concentration of biomolecules decreases along the longitudinal axisof the device from well region 13 a towards well region 14 a forming asurface concentration gradient of immobilized biomolecules and thebiological sample of cells potentially responds to this surfacegradient. This cell haptotaxis may be subsequently observed, measured,and recorded.

With respect to particular specifications of device 10, top member 11 ismade of a material that is adapted to effect conformal contact,preferably reversible conformal contact, with support member 16.According to embodiments of the present invention, the flexibility ofsuch a material, among other things, allows the top member toform-fittingly adhere to the upper surface U of support member 16 insuch a way as to form a substantially fluid-tight seal therewith. Theconformal contact should preferably be strong enough to prevent slippageof the top member on the support member surface. Where the conformalcontact is reversible, the top member may be made of a material havingthe structural integrity to allow the top member to be removed by asimple peeling process. This would allow top member 11 to be removed andcells at certain positions collected. Preferably, the peeling processdoes not disturb any surface treatment or cell positions of supportmember 16. Physical striations, pockets, SAMs, gels, peptides,antibodies, or carbohydrates can be placed on support member 16 and thetop member 11 subsequently can be placed over support member 16 withoutany damage to these structures. Additionally, the substantiallyfluid-tight seal effected between top member 11 and support member 16 byvirtue of the conformal contact of top member 11 with support member 16prevents fluid from leaking from one chamber to an adjacent chamber, andalso prevents contaminants from entering the wells. The seal preferablyoccurs essentially instantaneously without the necessity to maintainexternal pressure. The conformal contact obviates the need to use asealing agent to seal top member 11 to support member 16. Althoughembodiments of the present invention encompass use of a sealing agent,the fact that such a use is obviated according to a preferred embodimentprovides a cost-saving, time-saving alternative, and further eliminatesa risk of contamination of each chamber 12 by a sealing agent.Preferably, the top member 11 is made of a material that does notdegrade and is not easily damaged by virtue of being used in multipletests, and that affords considerable variability in the top member'sconfiguration during manufacture of the same. More preferably, thematerial may be selected for allowing the top member to be made usingphotolithography. In a preferred embodiment, the material comprises anelastomer such as silicone, natural or synthetic rubber, orpolyurethane. In a more preferred embodiment, the material ispolydimethylsiloxane (“PDMS”).

According to a preferred embodiment of a method of the presentinvention, standard photolithographic procedures can be used to producea silicon master that is the negative image of any desired configurationof top member 11. For example, the dimensions of chambers 12, such asthe size of well regions 13 a and 14 a, or the length of channel region15 a, can be altered to fit any advantageous specification. Once asuitable design for the master is chosen and the master is fabricatedaccording to such a design, the material is either spin cast, injected,or poured over the master and cured. Once the mold is created, thisprocess may be repeated as often as necessary. This process not onlyprovides great flexibility in the top member's design, it also allowsthe top members to be massively replicated. The present invention alsocontemplates different methods of fabricating device 10 including, forexample, e-beam lithography, laser-assisted etching, and transferprinting.

In another embodiment of the present invention, device 10 includes ahousing defining a chamber, the chamber including a first well regionincluding at least one first well; a second well region including atleast one second well; and a channel region including a plurality ofchannels connecting the first well region and the second well regionwith one another. The second well region is preferably horizontallyoffset with respect to the first well region is a test orientation ofthe device.

Device 10 preferably fits in the footprint of an industry standardmicrotiter plate. As such, device 10 preferably has the same outerdimensions and overall size of an industry standard microtiter plate.Additionally, when chamber 12 comprises a plurality of chambers, eitherthe chambers 12 themselves, or the wells of each chamber 12, may havethe same pitch of an industry standard microtiter plate. The term“pitch” used herein refers to the distance between respective verticalcenterlines between adjacent chambers or adjacent wells in the testorientation of the device. The embodiment of device 10, shown in FIG.1B, comprises 48 chambers designed in the format of a standard 96-wellplate, with each well fitting in the space of each macrowell of theplate. The size and number of the plurality of chambers 12 cancorrespond to the footprint of standard 24-, 96-, 384-, 768- and1536-well microtiter plates. For example, for a 96 well microtiterplate, device 10 may comprise 48 chambers 12 and therefore 48experiments can be conducted, and for a 384 well microtiter plate, thedevice may comprise 192 chambers 12, and therefore 192 experiments canbe conducted. The present invention also contemplates any other numberof chambers and/or wells disposed in any suitable configuration. Forexample, if pitch or footprint standards change or new applicationsdemand new dimensions, then device 10 may easily be changed to meetthese new dimensions. By conforming to the exact dimension andspecification of standard microtiter plates, embodiments of device 10would advantageously fit into existing infrastructure of fluid handling,storage, registration, and detection. Embodiments of device 10,therefore, may be conducive to high throughput screening as they mayallow robotic fluid handling and automated detection and data analysis.Top member 11 may additionally take on several different variations andembodiments. Depending on the test parameters, such as, for example,where chemotaxis, haptotaxis and/or chemoinvasion are to be monitored,the cell type, cell number, or distance over which chemotaxis orhaptotaxis is required, chamber 12 of top member 11 may have variousembodiments of which a few exemplary embodiments are discussed herein.With respect to a discrete chamber 12, the shape, dimensions, location,surface treatment, and numbers of channels in channel region 15 a andthe shape and number of wells 13 and 14 may vary.

Regarding the shape of channel region 15 a, each channel 15 in thechannel region 15 a is not limited to a particular cross-sectionalshape, as taken in a plane perpendicular to its longitudinal axis. Forexample, the cross section of any given channel 15 can be hexagonal,circular, semicircular, ellipsoidal, rectangular, square, or any otherpolygonal or curved shape.

Regarding the dimensions of a channel 15, the length L of a givenchannel 15 can vary based on various test parameters. For instance, thelength L of a given channel 15 may vary in relation to the distance overwhich chemotaxis or haptotaxis is required to occur. For example, thelength L of a given channel 15 can range from about 3 μm to about 18 mmin order to allow cells sufficient distance to travel and thereforesufficient opportunity to observe cell chemotaxis/haptotaxis andchemoinvasion. The width W and depth D of a given channel 15 may alsovary as a function of various test parameters. For examples, the width Wand depth D of a given channel 15 may vary, in a chemotaxis, haptotaxisand/or chemoinvasion device, depending on the size of the cell beingstudied and whether a gel matrix is added to the given channel 15.Generally, where the test device is a chemotaxis, haptotaxis and/orchemoinvasion device, a given channel 15's width W and depth D may beapproximately in the range of the diameter of the cell being assayed. Todiscount random cellular movement, at least one of the depth D or widthW of a given channel 15 should preferably be smaller than the diameterof the cell when no gel matrix is placed in the given channel 15 so thatwhen the cells are activated, they can “squeeze” themselves through thegiven channel toward the test agent. If a given channel 15 contains agel matrix, then, the depth D and width W of the given channel 15 may begreater than the diameter of the cell being assayed. Referring by way ofexample to the embodiments of FIGS. 1A-2C, if suspension cells such asleukocytes, which are about 3-12 μm in diameter, are in well 14 andchannel 15 contains no gel, then the width W of channel 15 should rangefrom about 3 microns to about 20 μm, and the depth D of channel 15should range from about 3 microns to about 20 μm but at least either thedepth D or width W of channel 15 should be smaller than the diameter ofthe cell. If leukocytes are in well 14 and channel 15 contains a gelmatrix, then the width W of channel 15 should range from about 20 toabout 100 μm and the depth D should range from about 20 μm to about 100μm, and both the width W and depth D of channel 15 can be greater thanthe diameter of the cell assayed. Similarly, if adherent cells, such asendothelial cells which are 3-10 microns in diameter before adherence,are in well 14 and channel 15 contains no gel, then the width W anddepth D of channel 15 can range from about 3 to about 20 μm, but atleast either the width W or depth D of channel 15 should be smaller thanthe diameter of the cell assayed. If adherent cells are in well 14 andchannel 15 contains a gel matrix then the width W and depth D of channel15 should range from about 20 μm to about 200 μm and both the width Wand depth D of channel 15 can be greater than the diameter of the cellassayed.

As seen in FIGS. 2A-2C channel 15 may connect the first well 13 to thesecond well 14 at respective sides of the wells, as shown in FIGS. 2Aand 2C or at a central region of the wells, as shown in FIG. 2B.

The number of channels in channel region 15 a between well regions 13 aand 14 a can also vary. Channel region 15 a may include a plurality ofchannels, as shown by way of example in FIGS. 3A-3C. As seen in FIG. 3A,in a preferred configuration, the length L of each channel 15 i-nbetween well 13 and well 14 is identical. In another embodiment as seenin FIG. 3B, the length L of each channel 15 i-15 n of channel region 15a increases in the direction of well 14, starting from channel 15 i inthe side portion 12 a of chamber 12 to channel 15 n in the side portion12 b of chamber 12. In one embodiment, as seen in FIG. 3B, the length Lof each successive channel in the plurality of channels 15 of chamber 12increases in a direction of a width W of the channels with respect to apreceding one of the plurality of channels such that respective channelinlets at one of the first well region and the second well region, suchas well region 13 a as shown, are aligned along the direction of thewidth W of the channels. Although, in this configuration, the cellstraveling in any particular channel will exit the channels and enterwell 14 at points longitudinally offset with respect to one another, thesection of channel region 15 a closest to well region 13 a is positionedso that cells ultimately entering the different channels will be alignedin a direction of the width W of the channels so that there is nolongitudinal offset between them. Therefore, in comparing two adjacentchannels, a first group of cells entering channel 15 i has an entryposition that is not longitudinally offset with respect to a seconddifferent group of cells entering channel 15 j, but the first group ofcells exiting channel 15 i has an exit point longitudinally offset fromthe second group of cells exiting channel 15 j. In a differentembodiment of the present invention illustrated in FIG. 3C, the width Wof each channel 15 i-15 n increases starting from channel 15 i in theside portion 12 a of chamber 12 to channel 15 n in the side portion 12 bof chamber 12. Preferably, the width W or depth D of each successivechannel of the plurality of channels increases in a direction of a widthW of the channels with respect to a preceding one of the plurality ofchannels. Alternatively, a depth D of each successive channel couldincrease (not shown) along a direction of the width W of the channels.It is understood to those skilled in the art, that various embodimentsaltering the dimensions of the channels in the channel region 15 a arewithin the scope of the present invention. For example, the length ofthe channels 15 i-15 n need not increase in a continuous manner fromchannel 15 i to 15 n as illustrated in FIG. 3B. Instead, channel 15 i-15n may have varying lengths following no particular order or pattern.

With respect to surface treatment of a given channel 15, to simulate invivo conditions where cells are surrounded by other cells, the lateralwalls of a given channel 15 may be coated with cells, such asendothelial cells 40 as seen in FIG. 4B. Non-limiting examples ofendothelial cells include human umbilical vein endothelial cells or highendothelial venule cells. In another embodiment, a given channel 15 isfilled with a gel matrix such as gelatin, agarose, collagen, fibrin, anynatural or synthetic extracellular proteinous matrix or basal membranemimic including, but not limited to MATRIGEL™ (Becton DickensonLabware), or ECM GEL, (Sigma, St. Louis, Mo.), or other hydrogelscontaining certain factors such as cytokines, growth factors,antibodies, ligands for cell surface receptors, or chemokines.Preferably, the gel has a substantially high water content and is porousenough to allow cell chemotaxis and invasion. As mentioned above, whenthe test agent comprising a soluble test substance is placed in well 13,the gel facilitates formation of a solution concentration gradient alongthe longitudinal axis of chamber 12. Additionally, adding a gel matrixto a given channel 15 simulates the natural environment in the body, asenzyme degradation through extracellular matrix is a crucial step in theinvasive process.

According to the present invention, the individual wells of each wellregion 13 a or 14 a may have any shape and size. For example, the topplan contour of a given well may be circular, as shown in FIGS. 1A-2C,or trapezoidal as shown in FIGS. 5 and 6. Alternatively, the top plancontour of a given chamber may be generally in the shape of a “figure 8”as shown in FIG. 7. Preferably when using a soluble test substance asthe test agent, the shape of well 13 is such that soluble test substanceis readily able to access the channel 15 and thereby form the necessarysolution concentration gradient along the length L of channel 15.Preferably, the shape of well 14 is such that cells are not deferred,detained, or hindered from migrating out of the first well 14, forexample, by accumulating in a corner, side or other dead space of well14. Although possible accumulation of cells in a dead space of well 14is not restricted to any particular cell number, there exists a greaterlikelihood of cells accumulating in a corner of well 14 if a largenumber of cells are used. Therefore to maximize accessibility to theconcentration gradient and to minimize the “wasting” of cells when alarge cell sample is utilized, it is important that the shape of well 14be such that a sufficiently high percentage of cells, particularly thecells in the area of well 14 furthest from channel 15, are capable ofmigrating out of well 14. In a different embodiment that also addressesthe problem of the wasting of cells, well 14 may be shaped such that allcells have a higher probability of accessing the concentration gradient.For example as seen in FIG. 8, the length L_(w) of well 14 in a top planview thereof is minimized to decrease the surface area of the well. Assuch, the cells are closer to the concentration gradient formed inchannel 15. In a preferred embodiment, the L_(w) of well 14 in a topplan view thereof is about 1 mm to about 2 mm.

In addition to variations of components of a discrete chamber 12, thepresent invention also contemplates variations in the overall chamber 12as well as variations from chamber to chamber. With respect to theoverall chamber 12, in one embodiment, the chambers 12 are sized so thata complete chamber 12 fits into the area normally required for a singlewell of a 96-well plate. In this configuration, 96 different assayscould be performed in a 96-well plate. In another embodiment, the 1:1ratio of a first well to second well, as present in the aforementionedembodiments, is altered by modifying chamber 12. For example as seen inFIG. 9, device 10 includes a chamber 12 having a first well region 13 ahaving a plurality of first wells 102, 103, 104 and 105 connected to oneanother, a second well region 14 a having a plurality of wells 106, 107,108 and 109, and a channel region 15 a having a plurality of channels 15connecting respective ones of the first wells to respective ones of thesecond wells. Each well of the first well region 13 a may receive thesame test agent, and each well of the second well region 14 a mayreceive a different cell type. Alternatively, each well of the firstwell region 13 a may receive a different test agent, and each well ofthe second well region 14 a may receive the same cell type. Thisconfiguration allows several different cell types or different testagents to be tested simultaneously. In an alternative embodiment as seenin FIG. 10, each channel 15 of channel region 15 a comprises subchannelsas shown. This arrangement not only allows several different cell typesor test agents to be tested simultaneously but also generates severaltests of each test agent or cell type.

FIG. 11 illustrates an alternative chamber configuration of a testdevice according to an alternative embodiment of the present invention.In this embodiment, chamber 12 comprises a first well region 13 aconnected by a channel region 15 a including a single channel 15 to asecond well region 14 a including a single well 14. The first wellregion contains a plurality of first wells, 17 a, 18 a, and 19 a and aplurality of capillaries, a first perimeter capillary 17, a centercapillary 18, and a second perimeter capillary 19 connected torespective ones of the plurality of first wells. All three of thecapillaries converge at a junction into channel 15, which is connectedwith the second well region 14 a. Well region 13 a is not limited tocontaining only three capillaries and can contain any number ofadditional capillaries (not shown). First wells 17 a-19 a may, forexample, be adapted to receive solutions of biomolecules, which areallowed to flow into channel 15 and adsorb nonspecifically to theregions of the surface over which the solution containing thebiomolecules flows. First wells 17 a-19 a are also adapted tosubsequently receive cells. With respect to variations from chamber tochamber, in one embodiment, the length L of each channel 15 increasesalong one or more dimensions of top member 11 from one chamber to theadjacent chamber. In an alternative embodiment, all chambers 12 havechannel 15 of the same length L. The width W of each channel 15 can alsovary and can increase along one or more dimensions of top member 11 fromone chamber to the adjacent chamber. In an alternative embodiment, allchambers 12 have channel 15 of the same width W. FIG. 4A is a top planview of an embodiment of the present invention where, within top member11, different chambers have various channel sizes and shapes, such sizesand shapes being in no particular order, pattern, or arrangement. Byemploying this varied configuration, the best channel region design fora given test may be obtained. In other words, where the optimal channelregion design is determined, a new assay plate configured solely tothose specifications may be employed.

Support member 16 of device 10 provides a support upon which top member11 rests and can be made of any material suitable for this function.Suitable materials are known in the art such as glass, polystyrene,polycarbonate, PMMA, polyacrylates, and other plastics. Where device 10is a chemotaxis, haptotaxis and/or chemoinvasion device, it ispreferable that support member 16 comprise a material that is compatiblewith cells that may be placed on the surface of support member 16.Suitable materials may include standard materials used in cell biology,such as glass, ceramics, metals, polystyrene, polycarbonate,polypropylene, as well as other plastics including polymeric thin films.A preferred material is glass with a thickness of about 0.1 to about 2mm, as this may allow the viewing of the cells with optical microscopytechniques.

Similar to top member 11, support member 16 can have several differentembodiments. In particular, the configuration and surface treatment ofsupport member 16 may vary.

As seen in a side view of support member 16 in FIG. 12, the uppersurface U of support member 16, which underlies top member 11, may besloped at predetermined regions thereof with respect to a horizontalplane at less than a 90° angle. In the shown embodiment, thepredetermined regions correspond to bottom surfaces of respective wells,surface 16 a corresponding to a bottom surface of a well 13, and surface16 b corresponding to a bottom surface of well 14. Surface 16 c, inturn, corresponds to a bottom surface of channel 15. In this embodiment,the given configuration facilitates suspended cells flowing in thedirection of the downward slope of top surface 16 b of support member 16to become more readily exposed to the concentration gradient. If asoluble test substance is used as the test agent in well 13 of device10, then top surface 16 a of support member 16 may also be downwardlysloped with respect to a horizontal plane at less than a 90° angle tofacilitate exposure of the test substance to channel 15 in order tofacilitate formation of the solution concentration gradient.

Support member 16 may also have a treatment on or embedded into itssurface. This treatment may serve numerous functions, including, forexample, facilitating the placement, adhesion or movement of cells beingstudied, and simulating in vivo conditions. Numerous surfaceconfigurations and chemicals may be used alone or in conjunction forthis treatment. For example, in one embodiment support member 16includes a patterned self-assembled monolayer (SAM) on a gold surface orother suitable material. SAMs are monolayers typically formed ofmolecules each having a functional group that selectively attaches to aparticular surface, the remainder of each molecule interacting withneighboring molecules in the monolayer to form a relatively orderedarray. By using SAMs, various controls of biological interactions may beemployed. For example, SAMs may be arrayed or modified with various“head groups” to produce “islands” of biospecific surfaces surrounded byareas of bio-inert head groups. Further, SAMs may be modified to have“switchable surfaces” that may be designed to capture a cell and then besubsequently modified to release the captured cell. Moreover, it mayalso be desirable to utilize a bioinert support member material toresist non-specific adsorption of cells, proteins, or any otherbiological material. Consequently, the use of SAMs on support member 16may be advantageous.

The present invention also contemplates, as seen in FIG. 13, the use ofany system known in the art to detect and analyze cell chemotaxis,haptotaxis, and chemoinvasion. In particular, the present inventioncontemplates the use of any system known in the art to visualize changesin cell morphology as cells move across channel 15, to measure thedistance cells travel in channel 15, and to quantify the number of cellsthat travel to particular points in channel 15. As such the presentinvention contemplates both “real-time” and “endpoint” analysis ofchemotaxis, haptotaxis, and chemoinvasion. In one embodiment, the device122 includes an observation system 120 and a controller 121. Thecontroller 121 is in communication with the observation system 120 vialine 122. The controller 121 and observation system 120 may bepositioned and programmed to observe, record, and analyze chemotaxis andchemoinvasion of the cells in the device. The observation system 120 maybe any of numerous systems, including a microscope, a high-speed videocamera, and an array of individual sensors. Nonlimiting examples ofmicroscopes include phase-contrast, fluorescence, luminescence,differential-interference-contrast, dark field, confocal laser-scanning,digital deconvolution, and video microscopes. Each of these embodimentsmay view or sense the movement and behavior of the cells before, during,and after the test agent is introduced. At the same time, theobservation system 120 may generate signals for the controller 121 tointerpret and analyze. This analysis can include determining thephysical movement of the cells over time as well as their change inshape, activity level or any other observable characteristic. In eachinstance, the conduct of the cells being studied may be observed in realtime, at a later time, or both. The observation system 120 andcontroller 121 may provide for real-time observation via a monitor. Theymay also provide for subsequent playback via a recording system of somekind either integrated with these components or coupled to them. Forexample, in one embodiment, cell behavior during the desired period ofobservation is recorded on VHS format videotape through a standard videocamera positioned in the vertical ocular tubes of a triocular compoundmicroscope or in the body of an inverted microscope and attached to ahigh quality video recorder. The video recorder is then played into adigitization means, e.g., PCI frame grabber, for the conversion ofanalog data to digital form. The electronic readable (digitized) data isthen accessed and processed by an appropriate dynamic image analysissystem, such as that disclosed in U.S. Pat. No. 5,655,028 expresslyincorporated in its entirety herein by reference. Such a system iscommercially available under the trademark DIAS® from Solltech Inc.(Oakland, Iowa). Software capable of assisting in discriminating cellsfrom debris and other detection artifacts that might be present in thesample should be particularly advantageous. In either case, thesecomponents may also analyze the cells as they progress through theirreaction to the test agent.

In one embodiment, the present invention contemplates the use of anautomated analysis system, as illustrated in FIG. 15, to analyze datameasuring the distance cells travel in channel 15, and to quantify thenumber of cells that travel to particular points in channel 15. FIG. 15is a block diagram of an automated analysis system 100 including, forexample, an image preprocessing stage 110, an object identificationstage 120 and a migration analysis stage 130. The image preprocessingstage 110 may receive digital image data of chamber 12 from a digitalcamera or other imaging apparatus as described above. The data typicallyincludes a plurality of image samples at various spatial locations(called, “pixels” for short) and may be provided as color or grayscaledata. The image preprocessing stage 110 may alter the captured imagedata to permit algorithms of the other stages to operate on it. Theobject identification stage 120 may identify objects from within theimage data. Various objects may be identified based on the test to beperformed. For example, the object identifier may identify channels 15,cells or cell groups from within the image data. The migration analysisstage 130 may perform the migration analysis designated for testing.FIG. 15 illustrates a number of blocks that may be included within theimage preprocessing stage 110. Essentially, the image preprocessingstage 110 counteracts image artifacts that may be present in thecaptured image data as a result of imperfections in the imager or thedevice. In one embodiment, the image preprocessing stage 110 may includean image equalization block 140. The equalization 140 may findapplication in embodiments where sample values of captured image data donot occupy the full quantization range available for the data. Forexample, an 8-bit grayscale system permits 256 different quantizationlevels for input data (0-255). Due to imperfections in the imagingprocess, it is possible that pixel values may be limited to a narrowrange, say the first 20 quantization levels (0-20). The equalization 140may re-scale sample values to ensure that they occupy the full rangeavailable in the 8-bit system.

In another embodiment, the equalization block 140 may re-scale samplevalues based on a color or wavelength. Conventional cellular analysistechniques often cause cells to appear in predetermined colors or withpredetermined wavelengths, which permits them to be distinguished fromother materials captured by the imager. For example, in fluorescentapplications, cells emit light at predetermined wavelengths. In nuclearstaining applications, cell nuclei are dyed with a material that causesthem to appear in the image data with predetermined colors. Theequalization block 140 may re-scale sample values having components thatcoincide with these expected colors or wavelengths. In so doing, theequalization block 140 effectively filters out other colors orwavelengths, a consequence that may be advantageous in later imageprocessing.

Image rotation is another image artifact that may occur from imperfectimaging apparatus. Although the channels 15 are likely to be generallyaligned with columns and rows of pixels in the image data, furtheranalysis may be facilitated if the alignment is improved. Accordingly,in an embodiment, the image preprocessing stage 110 may include an imagealignment block 150 that rotates the captured image data to counteractthis artifact. Once the rotation artifact has been removed from thecaptured image data, then image from individual channels 15 are likelyto coincide with a regular row or column array of pixel data. FIG. 16illustrates a method of operation for the image alignment block 150according to an embodiment of the present invention and described inconnection with exemplary image data illustrated in FIG. 17. In theexample of FIG. 17, channels 15 are aligned generally with rows of imagedata but for the rotation artifact. To counteract the rotation artifact,the image preprocessor may identify a band of image data coinciding witha boundary between second well 14 and the channels 15 themselves (block1010). In the case of FIG. 17, the band may constitute column 310.Generally, the area of second well 14 will be bright relative to thearea of channels 15 due the greater number of cells present therein.Thus, a histogram of image data values along a presumed direction of thechannels 15 may appear as shown in FIG. 18. The band 310 maybeidentified from an abrupt change in image data values along thisdirection.

Having identified a column of image data to be considered, the column310 may be split into two boundary boxes 320, 330 (block 1020). Bysumming the intensity of the image data in each of the two boundaryboxes and comparing summed values to each other, an orientation of therotation artifact may be determined (blocks 1030, 1040). In the exampleof FIG. 17, the rotation artifact causes more of second well 14 to fallwithin the area of boundary box 320 than of boundary box 330 (aclockwise artifact). The image data may be rotated counterclockwiseuntil the summed values of each boundary box 320, 330 become balanced.

Thus, if the image intensity of the first bounding box 320 is greaterthan that of the second bounding box 330, the image data may be rotatedin a first direction (block 1050). If the image intensity of the secondbounding box 330 is greater than that if the first bounding box 320, theimage data may be rotated in a second direction (block 1060). And whenthe image intensities are balanced, the method 1000 may conclude; therotation artifact has been corrected.

Returning to FIG. 15, the image preprocessing stage 110 also may processthe captured image data by cropping the image to the area occupied bychannels 15 themselves (block 160). As described, each test bed mayinclude a pair of wells interconnected by a plurality of channels. Formuch of the migration analysis, it is sufficient to measure cellularmovement or activity within channels 15 only. Activity in second well 14or the first well 13 need not be considered. In such an embodiment, theimage preprocessing stage 110 may crop the image data to remove pixelsthat lie outside channels 15.

The image preprocessing stage 110 also may include a thresholding block170, performing threshold detection upon the image data. Thethresholding block 170 may truncate to zero any sample having are-scaled value that fails to exceed a predetermined threshold. Suchthresholding is useful to remove noise from the captured image data. Inan embodiment, the thresholding block 170 may be integrated with theequalization block 140 discussed above. It need not be present as aseparate element. In some embodiments, particularly those where theequalization block 140 scales pixel values according to wavelengthcomponents, the thresholding block 170 may be omitted altogether. Anoutput of the image preprocessing stage 110 may be input to the objectidentification stage 120. The object identification stage 120 identifiesobjects from within the image data, including the channels themselvesand, optionally, individual cells. According to an embodiment, in afluorescent system, channels 15 may be identified by developing ahistogram of the fluorescent light along a major axis in the system(block 180). FIG. 19 illustrates image data that may have beendetermined from the example of FIG. 17. The major axis may coincide withthe boundary between the second well adapted to receive cells and thechannel region. Light intensity from within channel region 15 a area maybe summed along this axis, yielding a data set represented in FIG. 19.In a second stage, the data set is “dilated” (block 190). Dilation maybe achieved by applying a high pass filter to the data set or any otheranalogous technique. FIG. 20 illustrates the data set of FIG. 19 havingbeen subject to dilation.

From the data set of FIG. 20, the channels may be identified. Candidatechannel 15 positions may be identified to coincide with relativemaximums of the data set. Alternatively, candidate positions ofboundaries between channels 15 may be determined from relative minimumsfrom within the data set of FIG. 20. A final set of channel 15 positionsmay be determined from a set of parameters known about channel region 15a itself. For example, if channels 15 are known to have been providedwith a regular spacing among channels 15, any candidate channel 15position that would violate the spacing can be eliminated fromconsideration.

Returning to FIG. 15, in addition to identifying channels 15, individualcells may be identified within the image data (block 200). In anapplication where cells are marked with nuclear staining, identificationof individual cells merely requires an image processor to identify andcount the number of marked nuclei. The nuclei appear as a number of dotsof a predetermined color. In an application using fluorescing cells,identification of individual cells becomes more complicated. Individualcells can be identified relatively easily; they appear as objects ofrelatively uniform area in the image data. Identifying a number of cellsclustered together becomes more difficult. In this case, the number ofcells may be determined from the area or radius of the cluster in theimage data. The cluster is likely to appear in the image having somearea or cluster radius. By comparing the cluster's area or radius to thearea or radius of an individual cell, the number of cells may beinterpolated. Of course, identification of individual cells may beomitted depending upon the requirements of the migration analysis.

The final stage in the image processing system is the migration analysis130 itself. In one embodiment, coordinate data of each cell in thechannels 15 may be gathered and recorded. However, some testing need notbe so complicated. In a first embodiment, it may be sufficient merely toidentify the number of cells present in channel 15. In this case,identification of individual cells may be avoided by merely summingquantities of fluorescent light detected in each channel 15. From thismeasurement, the number of cells may be derived without investing theprocessing expense of identifying individual cells. The foregoingdescription presents image analysis that is relevant to a single channel15 to be tested. Of course, depending upon the requirements of themigration analysis 130, it may be desired to generate image samples of anumber of different channels 15. Further, it may be desirable togenerate image samples of a single channel 15 at different times. Theimage processing described above may be repeated for different channels15 and different times to accommodate for such test scenarios.

According to an embodiment, the image processing may account formanufacturing defects of individual channels 15. During imageprocessing, manufacturing defects may prevent cell migrations into achannel 15. In an embodiment, when the system 100 counts a number ofcells in the channel 15 (or derives the number from identified celllocations), it may compare the number to an expectation threshold. Ifthe number is below the expectation threshold, the system 100 mayexclude the channel 15 from migration analysis. In practice, thisexpectation threshold may be established as a minimum number of cellsthat are likely to enter a properly configured cell given the testconditions being analyzed under the migration analysis. If the actualnumber of cells falls below this threshold, it may lead to a conclusionthat channel 15 blocking conditions may be present.

The foregoing operations and processes of the analysis system 100 may beperformed by general purpose processing apparatus, such as computers,workstations or servers, executing software. Alternatively, some of theoperations or processes may be provided in a digital signal processor orapplication specific integrated circuit (colloquially, an “ASIC”).Additionally, these operations and processes, particularly thoseassociated with image preprocessing, may be distributed in processors ofa digital microscope system. Such variations are fully within the scopeof the present invention.

The present invention also contemplates the use of the aforementionedembodiments of device 10 to assay various elements of chemotaxis,haptotaxis and chemoinvasion. In general, the present invention providesfor a first assay comprising high throughput screening of test agents todetermine whether they influence chemotaxis, haptotaxis, andchemoinvasion. Test agents generally comprise either soluble testsubstances or immobilized test biomolecules and are generally placed infirst well region 13 a of chamber 12 of device 10. After determiningwhich test agents influence chemotaxis, by acting as chemoattractantsand promoting or initiating chemotaxis, by acting as chemorepellants andrepelling chemotaxis, or by acting as inhibitors and halting orinhibiting chemotaxis, then a second assay can be performed screeningtest compounds. The test compounds generally comprise therapeutics orchemotaxis/haptotaxis inhibitors and are generally introduced in secondwell region 14 a, which contains a biological sample of cells. The testcompounds are screened to determine if and how they influence the cells'chemotaxis or haptotaxis in response to the test agents.

In particular, a chemotaxis/haptotaxis and/or chemoinvasion assayaccording to an embodiment of the present invention involves a device 10including a housing comprising a top member 11 mounted to a supportmember 16. The top member and the support member are configured suchthat they together define a discrete assay chamber 12. The discreteassay chamber 12 includes a first well region 13 a connected by achannel 15 to a second well region 14 a. The first well region 13 aincludes at least one first well 13, each of the at least one first well13 being adapted to receive a test agent therein. The second well region14 a includes at least one second well 14 horizontally offset withrespect to the first well region 13 a in a test orientation of thedevice, each of the at least one second well 14 being adapted to receivea cell sample therein. Channel 15 includes at least one channelconnecting the first well region 13 a and the second well region 14 a toone another. The test agent received in first well 13 is a soluble testsubstance and/or immobilized test biomolecules. When the test agentcomprises immobilized test biomolecules, the biomolecules areimmobilized on an upper surface U of support member 16 constituting thebottom surface of well region 13 a as well as on upper surface U ofsupport member 16 constituting the bottom surface of channel region 15a.

Nonlimiting examples of biological samples of cells include lymphocytes,monocytes, leukocytes, macrophages, mast cells, T-cells, B-cells,neutrophils, basophils, eosinophils, fibroblasts, endothelial cells,epithelial cells, neurons, tumor cells, motile gametes, motile forms ofbacteria, and fungi, cells involved in metastasis, and any other typesof cells involved in response to inflammation, injury, or infection.Well region 14 a may receive only one cell type or any combination ofthe above-referenced exemplary cell types. For example, as describedabove, it is often desirable to provide a mixed cell population to moreeffectively create an environment similar to in vivo conditions. Wellregion 14 a may also receive cells at a particular cell cycle phase. Forexample, well region 14 a may receive lymphocytes in G₁ phase or G₀phase.

Nonlimiting examples of soluble test substances includechemoattractants, chemorepellants, or chemotactic inhibitors. Asexplained above, chemoattractants are chemotactic substances thatattract cells and once placed in well region 14 a, cause cells tomigrate towards well region 14 a. Chemorepellents are chemotacticsubstances that repel cells and once placed in well region 14 a, causecells to migrate away from well region 14 a. Chemotactic inhibitors arechemotactic substances that inhibit or stop chemotaxis and once placedin well region 14 a, cause cells to have inhibited migration or nomigration from well region 14 a. Non-limiting examples ofchemoattractants include hormones such as T₃ and T₄, epinephrine andvasopressin; immunological agents such as interleukein-2, epidermalgrowth factor and monoclonal antibodies; growth factors; peptides; smallmolecules; and cells. Cells may act as chemoattractants by releasingchemotactic factors. For example, in one embodiment, a sample includingcancer cells may be added to well 13. A sample including a differentcell type may be added to well 14. As the cancer cells grow they mayrelease factors that act as chemoattractants attracting the cells inwell 14 to migrate towards well 13. In another embodiment, endothelialcells are added to well 13 and activated by adding a chemoattractantsuch as TNF-α or IL-1 to well 13. Leukocytes are added to well 14 andmay be attracted to the endothelial cells in well 14.

Non-limiting examples of chemorepellants include irritants such asbenzalkonium chloride, propylene glycol, methanol, acetone, sodiumdodecyl sulfate, hydrogen peroxide, 1-butanol, ethanol, anddimethylsulfoxide; and toxins such as cyanide, carbonylcyanidechlorophenylhydrazone, endotoxins and bacterial lipopolysaccharides;viruses; pathogens; and pyrogens.

Nonlimiting examples of immobilized biomolecules includechemoattractants, chemorepellants, and chemotactic inhibitors asdescribed above. Further non-limiting examples of immobilizedchemoattactants include chemokines, cytokines, and small molecules.Further non-limiting examples of chemoattractants include IL-8, GCP-2,GRO-α, GRO-β, MGSA-β, MGSA-γ, PF₄, ENA-78, GCP-2, NAP-2, IL-8, IP10,I-309, I-TAC, SDF-1, BLC, BRAK, bolekine, ELC, LKTN-1, SCM-1β, MIG,MCAF, LD7α, eotaxin, IP-110, HCC-1, HCC-2, Lkn-1, HCC-4, LARC, LEC,DC-CK1, PARC, AMAC-1, MIP-2β, ELC, exodus-3, ARC, exodus-1, 6Ckine,exodus 2, STCP-1, MPIF-1, MPIF-2, Eotaxin-2, TECK, Eotaxin-3, ILC, ITAC,BCA-1, MIP-1α, MIP-1β, MIP-3α, MIP-3β, MCP-1, MCP-2, MCP-3, MCP-4,MCP-5, RANTES, eotaxin-1, eotaxin-2, TARC, MDC, TECK, CTACK, SLC,lymphotactin, and fractalkine; and other cells. Further non-limitingexamples of chemorepellants include receptor agonists and other cells.

In order to perform a test, such as a chemotaxis and/or chemoinvasionassay utilizing a soluble test substance, the test device 10 is firstfabricated. A preferred embodiment of the method of making the deviceaccording to the present invention will now be described. A master thatis the negative of top member 11 is fabricated by standardphotolithographic procedures. A predetermined material is spin coated orinjection molded onto the master. The predetermined material is thencured, peeled off the master to comprise top member 11 and placed ontosupport member 16.

Where the test device 10 is a chemotaxis, haptotaxis and/orchemoinvasion device, a rigid frame with the standard microtiterfootprint is preferably placed around the outer perimeter of top member11. In one embodiment, a gel matrix is poured into well region 13 a andallowed to flow into channel region 15 a. After the gel matrix sets,excess gel is removed from well regions 13 a and 14 a. In anotherembodiment, no gel matrix is added to channel region 15 a. Subsequently,a biological sample of cells is placed in well region 14 a and a testsubstance is placed in well region 13 a. In one embodiment, a lowconcentration of a test substance is placed in well region 14 a in orderto activate the cells and expedite the beginning of the assay.Alternatively, depending on the cells being studied and the soluble testsubstance being used, the soluble test substance may be introducedduring or after the cells have been placed in well region 14 a. Once thesoluble test substance has been introduced, by the process of diffusion,a solution concentration gradient of the test substance forms along thelongitudinal axis of channel region 15 a from well region 13 acontaining the test agent towards well region 14 a containing thebiological sample of cells. A secondary effect of this solution gradientis the formation of a physisorbed (immobilized) gradient. When thissolution gradient is established, some fraction of the solute of thetest substance may adsorb onto support member 16. This adsorbed layer oftest solute may also contribute to chemotaxis and chemoinvasion. Thebiological sample of cells may respond to this concentration gradientand migrate towards the higher concentration of the test substance,migrate away from the higher concentration of the test substance, orexhibit inhibited movement in response to the higher concentration ofthe test substance. It is through this chemotaxis in response to thegradient, that the chemotactic influence of the chemotactic substancecan be measured. Chemotaxis is assayed by measuring the distance thecells travel and the amount of time the cells take to reach apredetermined point in the channel region 15 a or the distance the cellstravel and the amount of time the cells take to reach a certain point inwell region 14 a (in the case of a chemorepellant that causes cells tomove away from the chemotactic substance).

Utilizing an alternative embodiment of device 10 containing analternative design of chamber 12, a solution concentration gradient isformed using a network of microfluidic channel regions. In thisembodiment as seen in FIG. 14, first well region well region 13 a ofchamber 12 has first wells, 20, 24, and 22, connected by a network ofmicrofluidic capillaries 23 to channel 15. In particular, first wellregion 13 a includes a plurality of first wells connected by a pluralityof capillaries 28 connected to respective ones of the plurality of firstwells and a plurality of subcapillaries 25 branched off such that eachof the plurality of subcapillaries is connected to each of the pluralityof capillaries at one end thereof and to channel 15 at another endthereof. Each first well, 20, 24, and 22 receives a differentconcentration of soluble test substance. After the three first wells,20, 24, and 22 are simultaneously infused with the three differentconcentrations of soluble test substance, the solution streams traveldown the network of channel regions, continuously splitting, mixing andrecombining. After several generations of branched subcapillaries, eachsubcapillary containing different proportions of soluble test substancesare merged into a single channel 15, forming a concentration gradientacross channel 15, perpendicular to the flow direction.

According to one embodiment of the present invention, biomolecules areimmobilized onto support member 16, preferably on the portion of uppersurface U constituting the bottom surface of channel 15 and of wellregion 13 a in any one of the embodiments of the test device of thepresent invention, such as the embodiments shown in FIGS. 1A-14. Theconcentration of biomolecules increases or decreases along thelongitudinal axis of the device from the upper surface of support member16 constituting the bottom surface of well region 13 a towards the uppersurface U of support member 16 constituting the bottom surface of wellregion 14 a thus forming a surface gradient. After the test biomoleculesare immobilized on support member 16, the top member is placed ontosupport member 16 and a rigid frame with the standard microtiterfootprint is placed around the outer perimeter of top member 11 andcells are added to well region 14 a. In an alternative embodiment, afterthe test biomolecules are immobilized on support member 16 and the topmember is placed over support member 16, a gel matrix is added tochannel region 15 a. Cells are subsequently added to well region 14 a.The biological sample of cells potentially respond to the concentrationgradient of immobilized biomolecules and migrates towards the higherconcentrations of the test biomolecules, away from the higherconcentrations of the test biomolecules, or exhibit inhibited migrationin response to the higher concentrations of the test biomolecules. Thesurface gradient can increase linearly or as a squared, cubed, orlogarithmic function or in any surface profile that can be approximatedin steps up or down.

The test biomolecules can be attached to and form surface gradients onthe upper surface U of support member 16 by various specific ornon-specific approaches known in the art as described in K. Efimenko andJ. Genzer, “How to Prepare Tunable Planar Molecular Chemical Gradient,”13 Applied Materials, 2001, No. 20, October 16; U.S. Pat. No. 5,514,incorporated herein by reference. For example, microcontact printingtechniques, or any other method known in the art, can be used toimmobilize on upper surface U of support member 16 a layer of SAMspresenting hexadecanethiol. Support member 16 is then exposed to highenergy light through a photolithographic mask of the desired gradientmicropattern or a grayscale mask with continuous gradations from whiteto black. When the mask is removed, a surface gradient of SAMspresenting hexandecanethiol remains. Support member 16 is then immersedin a solution of ethylene glycol terminated alkanethiol. The regions ofsupport member 16 with SAMs presenting hexadecanethiol will rapidlyadsorb biomolecules and the regions of the support member with SAMspresenting oligomers of the ethylene glycol group will resist adsorptionof protein. Support member 16 is then immersed in a solution of thedesired test biomolecules and the biomolecules rapidly adsorb only tothe regions of support member 16 containing SAMs presentinghexadecanethiol creating a surface gradient of immobilized biomolecules.

In another embodiment, the test biomolecules are immobilized on thesupport member 16 and a surface concentration gradient forms after thetop member 11 has been placed over support member 16 in any one of theembodiments of the test device of the present invention, such as theembodiments shown in FIGS. 1A-14. In this embodiment, discreteconcentrations of solution containing test biomolecules areconsecutively placed in well region 14 a and allowed to adsorbnon-specifically to support member 16. For example, first, a 1milligram/milliliter (mg/ml) of solution can first be placed in wellregion 14 a; second, a 1 microgram/milliliter (μg/ml) solution can beplaced in well region 14 a; last, a 1 nanogram/milliliter (ng/ml)solution of test biomolecules can be placed in well region 14 a. Thediffering concentrations of test biomolecules in solution result indiffering amounts of adsorption on support member 16.

Utilizing an alternative embodiment of device 10 containing analternative design of chamber 12 as seen in FIG. 11, an immobilizedbiomolecular surface gradient is formed based on the concept of laminarflow of multiple parallel liquid streams, a method known in the art.Based on this concept, when two or more streams with low Reynoldsnumbers are joined into a single stream, also with a low Reynoldsnumber, the combined streams flow parallel to each other withoutturbulent mixing. According to one embodiment, a solution of chemotacticbiomolecules is placed in 17 a and 19 a and a protein solution is placedin 18 a. The solutions are allowed to flow into channel region 15 aunder the influence of gentle aspiration at well region 14 a.Biomolecules adsorb nonspecifically to the regions of the surface overwhich the solution containing the biomolecules flows forming a surfacegradient. The wells are then filled with a suspension of cells andpotential haptotaxis of the cells towards the increasing concentrationgradient of biomolecules is observed and monitored. See generally, S.Takayama et al., “Patterning Cells and their Environment Using MultipleLaminar Fluid Flows in Capillary Networks” Pro. Natl. Acad. Sci. USA,Vol. 96, pp. 5545-5548, May 1999.

The present invention also contemplates an assay using both a solubleand surface gradient to determine whether the soluble test substance orthe immobilized test biomolecules more heavily influence chemotaxis andchemoinvasion. In this embodiment, an assay is performed by forming asurface gradient as described above, an assay is performed by forming asolution gradient as described above, an assay is performed by formingboth types of gradients and the results of all three assays arecompared. With respect to the combined gradient assay, test biomoleculesare immobilized on the upper surface U of support member 16 constitutingthe bottom surface of well region 13 a and on the upper surface ofsupport member 16 underlying channel region 15 a and the concentrationof biomolecules decreases along the longitudinal axis of chamber 12 fromwell region 13 a to well region 14 a, in any one of the embodiments ofthe test device of the present invention, such as the embodiments shownin FIGS. 1A-14. Additionally, a soluble test substance is added to wellregion 13 a. Such an embodiment creates surface and soluble chemotacticconcentration gradients that decrease in the same direction. If thecombined concentration gradients have a synergistic effect on chemotaxisand/or chemoinvasion, then both gradients should be used in screeningboth the cell receptor binding the chemotactic ligands of the solublechemotactic substance and the cell receptor binding the immobilizedbiomolecules. Both types of receptors are identified as important andtherapeutic agents that target both these receptors or a combination oftherapeutic agents, one targeting one receptor and another targeting theother receptor can be screened. If the combined concentration gradientsdo not have a synergistic effect, then the individual gradient that morestrongly promotes chemotaxis and/or chemoinvasion can be identified andthe cell receptor that binds to the chemotactic ligands of the testagent forming the gradient can be targeted.

Identifying optimal chemotactic ligand and receptor pairs is importantin understanding the biological pathways implicated in chemotaxis and/orchemoinvasion and developing therapeutic agents that target thesepathways. Accordingly, the present invention generally provides usingchemotactic test agents to determine which chemotactic receptorsexpressed on a cell's surface most heavily influence chemotaxis and/orchemoinvasion. In one embodiment, the present invention provides forhigh throughput screening of a class of chemoattractants known toattract a particular cell type having a receptor on the cell's surfacefor each chemoattractant within this class in order to identify whichreceptor is more strongly implicated in the chemotaxis and/orchemoinvasion process. After identifying this receptor, the presentinvention contemplates high-throughput screening of therapeutic agentsthat potentially block this receptor or bind to this receptor, dependingon whether chemotaxis and/or chemoinvasion is desired to be promoted orprevented. In another embodiment, the present invention provides forhigh throughput screening of different chemoattractants known to bind tothe same receptor on a particular cell type's surface, in order todetermine which chemoattractant ligand/receptor pair more heavilyinfluences chemotaxis and/or chemoinvasion. After identifying thisligand/receptor pair, the present invention contemplates high throughputscreening of therapeutic agents that target this receptor and eitherblock or activate this receptor depending one whether chemotaxis and/orchemoinvasion is desired to be promoted or prevented.

The present invention also contemplates high-throughput screening of aclass of chemotactic inhibitors known to inhibit chemotaxis of aparticular cell type having various chemotactic receptors on the cell'ssurface in order identify which receptor is more strongly implicated inthe chemotaxis and chemoinvasion process. After identifying thisreceptor, the present invention provides for high throughput screeningof therapeutic agents that potentially block this receptor as well (ifsuch action is desired).

In one embodiment of the present invention, an assay is performed todetermine whether a test compound inhibits cancer cell invasion. In thisembodiment, untreated cancer cells are placed in well region 14 a and atest agent is placed in well region 13 a of chamber 12 in any one of theembodiments of the test device of the present invention, such as theembodiments shown in FIGS. 1A-14. Cell chemotaxis and invasion ismeasured and recorded. After a suitable test agent is identified (onethat chemically attracts the cancer cells) another assay is run inchamber 12. In this subsequent assay, cancer cells are placed in wellregion 14 a and a test compound, for example, a therapeutic, is alsoplaced in well region 14 a. In another embodiment, the test compound isalso placed in channel region 15 a. If a gel matrix is to be added tochannel region 15 a, the test compound can be mixed with the gel matrixbefore the gel is contacted with channel region 15 a during fabricationof device 10. A subsequent sample of the test agent identified in thefirst assay is placed in well region 13 a and the chemotaxis andinvasion of the cells treated with the test compound is compared to thechemotaxis and invasion of the cells not treated with the test compound.The test compound's anti-cancer potential is measured by whether thetreated cancer cells have a slower chemotaxis and invasion rate than theuntreated cancer cells.

With respect to another exemplary use of the chemotaxis andchemoinvasion device of the present invention, the device can be used toassay cells' response to the inflammatory response. A local infection orinjury in any tissue of the body attracts leukocytes into the damagedtissue as part of the inflammatory response. The inflammatory responseis mediated by a variety of signaling molecules produced within thedamaged tissue site by mast cells, platelets, nerve endings andleukocytes. Some of these mediators act on capillary endothelial cells,causing them to loosen their attachments to their neighboringendothelial cells so that the capillary becomes more permeable. Theendothelial cells are also stimulated to express cell-surface moleculesthat recognize specific carbohydrates that are present on the surface ofleukocytes in the blood and cause these leukocytes to adhere to theendothelial cells. Other mediators released from the damaged tissue actas chemoattractants, causing the bound leukocytes to migrate between thecapillary endothelial cells into the damaged tissue. To study leukocytechemotaxis, in one embodiment, channel region 15 a is treated tosimulate conditions in a human blood capillary during the inflammatoryresponse. For example, the side walls of channel region 15 a are coatedwith endothelial cells expressing cell surface molecules such asselections, for example as shown in FIG. 4B. Leukocytes are then addedto well region 14 a and a known chemoattractant is added to well region13 a in any one of the embodiments of the test device of the presentinvention, such as the embodiments shown in FIGS. 1A-14. Other suitablecell types that can be added to well region 14 a are neutrophils,monocytes, T and B lymphocytes, macrophages or other cell types involvedin response to injury or inflammation. The leukocytes' chemotaxis acrosschannel region 15 a towards well region 13 a is observed. Depending onthe type of infection to be studied, different categories of leukocytescan be used. For example, in one embodiment studying cell chemotaxis inresponse to a bacterial infection, well region 14 a receivesneutrophils. In another embodiment studying cell chemotaxis in responseto a viral infection, well region 14 a receives T-cells. In anotherembodiment simulating the process of angiogenesis, it is known in theart that growth factors applied to the cornea induce the growth of newblood vessels from the rim of highly vascularized tissue surrounding thecornea towards the sparsely vascularized center of the cornea. Thereforein another exemplary assay utilizing the chemotaxis and chemoinvasiondevice, cells from corneal tissue are placed in well region 13 a andendothelial cells are placed in well region 14 a in any one of theembodiments of the test device of the present invention, such as theembodiments shown in FIGS. 1A-14. A growth factor is added to wellregion 13 a and chemotaxis of the endothelial cells is observed,measured and recorded. Alternatively, since angiogenesis is alsoimportant in tumor growth (in order to supply oxygen and nutrients tothe tumor mass), instead of adding growth factor to well region 13 a,cancer cells from corneal tissue that produce angiogenic factors such asvascular endothelial growth factor (VEGF) could be added to well region13 a and normal endothelial cells added to well region 14 a. In adifferent embodiment also related to the study of angiogenesis, mastcells, macrophages, and fat cells that release fibroblast growth factorduring tissue repair, inflammation, and tissue growth are placed in wellregion 13 a and endothelial cells are placed in well region 14 a. Sinceduring angiogenesis, a capillary sprout grows into surroundingconnective tissue, to further simulate conditions in vivo, channelregion 15 a can be filled with a gel matrix.

There are several variations and embodiments of the aforementionedassays. One embodiment involves the number of channels connecting wellregion 13 a and well region 14 a of chamber 12 of device 10. In oneembodiment, such as the ones shown in FIGS. 3A-3C, there are multiplechannels connecting well region 13 a to well region 14 a. By usingmultiple channels, multiple assays can be performed simultaneously usingone biological sample of cells. In such an embodiment, all assays areperformed under uniform and consistent conditions and therefore providestatistically more accurate results. For example, each assay begins withexactly the same number of potentially migratory cells and exactly thesame concentration of test agent. Once a concentration gradient forms,each assay is exposed to the gradient for the same period of time. Thesemultiple channels also provide redundancy in case of failure in theassay.

Another embodiment of the cell invasion and chemotaxis assay of thepresent invention involves the placement of cells in well region 14 a ofchamber 12 in any one of the embodiments of the test device of thepresent invention, such as the embodiments shown in FIGS. 1A-14. Thecells may be patterned in a specific array on the upper surface U ofsupport member 16 constituting the bottom surface of well region 14 a ormay simply be deposited in no specific pattern or arrangement in wellregion 14 a. If the cells are patterned in a specific array on the uppersurface of support member 16 constituting the bottom surface of wellregion 14 a, then preferably, during the fabrication of device 10, theupper surface of support member 16 constituting the bottom surface ofwell region 14 a is first patterned with cells and then top member 11 isplaced over support member 16. It is desirable to monitor cellularmovement from a predetermined “starting” position to accurately measurethe distance and time periods the cells travel. As such, in oneembodiment, the cells are immobilized or patterned upon the supportmember underlying the first well in such a manner that the cells'viability is maintained and their position is definable so thatchemotaxis and invasion may be observed. There are several techniquesknown in the art to immobilize and pattern the cells into discreetarrays onto the support member. A preferred technique is described incopending application Ser. No. 60/330,456. In one embodiment, a cellposition patterning member is used to pattern the cells into definableareas onto the upper surface U of support member 16 constituting thebottom surface of well region 14 a of top member 11.

If, for example, top member 11 is fabricated in the footprint of astandard 96-well microtiter plate such that wells 13 and 14 correspondto the size and shape of the macrowells of the microtiter plate (notshown), then the cell position pattern member has outlined areas whichcorrespond to the size and shape of wells 13 and 14 and thereforecorrespond to the size and shape of the macrowells of the microtiterplate. Each outlined area has micro through holes through which thecells will be patterned. In order to pattern the cells, the cellposition patterning member is contacted with support member 16 and theoutlined areas of the cell position patterning member are aligned withportion of upper surface U of support member 16 that constitutes thebottom surface of well region 14 a, and will ultimately correspond towell region 14 a once top member 11 is contacted with support member 16.Cells are then deposited over the cell position patterning member andfilter through the micro through holes of the patterning member onto thesupport member underlying the areas corresponding to through-holescorresponding to second well regions 14 a of chambers 12. Top member 11is then placed over support member 16 such that through-holes 14 a areplaced over the area of support member 16 in which the cells arepatterned. These patterning steps result in discrete arrays of cells inwell region 14 a.

Preferably, the cell position patterning member comprises an elastomericmaterial such as PDMS. Using PDMS for the patterning member provides asubstantially fluid-tight seal between the patterning member and thesupport member. This substantially fluid-tight seal is preferablebetween these two components because cells placed in the wells are lesslikely to infiltrate adjoining wells if such a seal exists between thepatterning member and the support member. The arrangement of the microthrough holes of the patterning member may be rectangular, hexagonal, oranother array resulting in the cells being patterned in these respectiveshapes. The width of each micro-through hole may be varied according tocell types and desired number of cells to be patterned. For example, ifthe width of both cell and micro through hole is 10 microns, only onecell will deposit through each micro through hole. Thus, in thisexample, if the width of micro through hole is 100 microns up toapproximately 100 cells may be deposited.

The present invention also contemplates the patterning of more than onecell type on the upper surface of support member 16 constituting thebottom surface of well region 14 a in any one of the embodiments of thetest device of the present invention, such as the embodiments shown inFIGS. 1A-14. Since cells of one type in vivo rarely exist in isolationand are instead in contact and communication with other cell types, itis desirable to have a system in which cells can be assayed in anenvironment more like that of the body. For example, since cancer cellsare never found in isolation, but rather surrounded by normal cells, anassay designed to test the effect of a drug on cancer cells would bemore accurate if the cancer cells in the assay were surrounded by normalcells. In testing an anti-cancer drug, cancer cells may be patterned onthe upper surface of support member 16 constituting the bottom surfaceof well region 14 a in any given one of the embodiments of the testdevice of the present invention, such as the embodiments of FIGS. 1A-14,and then through a separate patterning procedure, the cancer cells maybe surrounded by stromal cells. To pattern two different cell types onthe upper surface of support member 16 constituting the bottom surfaceof well region 14 a, a micro cell position patterning member, asdescribed above, is contacted with support member 16 and the outlinedareas of the cell position patterning member are aligned with theportion of upper surface U of support member 16 that constitutes thebottom surface of well region 14 a, and will ultimately correspond towell region 14 a once top member 11 is contacted with support member 16.Cells of a first type may then be deposited over the cell positionpatterning member and filter through the micro through holes of thepatterning member onto the portion of the upper surface U of supportmember 16 constituting the bottom surface of well region 14 a. The microcell position patterning member may then be removed from support member16. A macro cell position patterning member with outlined areas thatcorrespond to the size and shape of wells 13 and 14 and may thereforecorrespond to the size and shape of the macrowells of a 96 wellmicrotiter plate. The macro cell position patterning member has macrothrough holes. A macro through hole of the macro cell positionpatterning member encompasses an area larger than the surface areadefined by a micro through hole of the micro cell position patterningmember, but smaller than the surface area defined by well region 14 a ofchamber 12. The macro cell position patterning member may then becontacted with support member 16. Cells of a second type may then bedeposited over the macro cell position patterning member and filterthrough the macro through holes of the macro cell position patterningmember onto the portion of upper surface U of support member 16constituting the bottom surface of well regions 14 a once top member 11is contacted with support member 16. Such patterning arrangement mayresult in cells of a second type surrounding and “stacking” cells of afirst type. If it is desired to only have the cells of the second typestack the cells of the first type, then the same micro cell positionpatterning member used to deposit the first cell type or a differentmicro cell position patterning member having the exact sameconfiguration as the patterning member used to deposit cells of a firsttype, may be used to deposit cells of a second type. After the cells arepatterned on support member 16, top member 11 may be contacted withsupport member 16 such that through holes in top member 11 correspondingto the well region 14 a encompass the areas patterned with cells. Thisessentially results in cells being immobilized in a specific arraywithin well region 14 a.

Notwithstanding how many different cell types are patterned on the uppersurface of ti support member 16 constituting the bottom surface of wellregion 14 a, the cells may be patterned on the support member throughseveral methods known in the art. For example, the cells may bepatterned on support member 16 through the use of SAMS. There areseveral techniques known in the art to pattern cells through the use ofSAMs of which a few exemplary techniques disclosed in U.S. Pat. No.5,512,131 to Kumar et al., U.S. Pat. No. 5,620,850 to Bambad et al.,U.S. Pat. No. 5,721,131 to Rudolph et al., U.S. Pat. Nos. 5,776,748 and5,976,826 to Singhvi et al. are incorporated by reference herein.

Several methods are known in the art to tag the cells in order toobserve and measure the aforementioned parameters. In one embodiment, anunpurified sample containing a cell type of interest is incubated with astaining agent that is differentially absorbed by the various celltypes. The cells are then placed in well region 14 a of chamber 12 inany given one of the embodiments of the test device of the presentinvention, such as the embodiments of FIGS. 1A-14. Individual, stainedcells are then detected based upon color or intensity contrast, usingany suitable microscopy technique(s), and such cells are assignedpositional coordinates. In another embodiment, an unpurified cell sampleis incubated with one or more detectable reporters, each reportercapable of selectively binding to a specific cell type of interest andimparting a characteristic fluorescence to all labeled cells. The sampleis then placed in well region 14 a of chamber 12 in any given one of theembodiments of the test device of the present invention, such as theembodiments of FIGS. 1A-14. The sample is then irradiated with theappropriate wavelength light and fluorescing cells are detected andassigned positional coordinates. One skilled in the art will recognizethat a variety of methods for discriminating selected cells from othercomponents in an unpurified sample are available. For example, thesemethods can include dyes, radioisotopes, fluorescers, chemiluminescers,beads, enzymes, and antibodies. Specific labeling of cell types can beaccomplished, for example, utilizing fluorescently-labeled antibodies.The process of labeling cells is well known in the art as is the varietyof fluorescent dyes that may be used for labeling particular cell types.

Cells of a chosen type may be also differentiated in a mixed-cellpopulation, for example, using a detectable reporter or a selectedcombination of detectable reporters that selectively and/orpreferentially bind to such cells. Labeling may be accomplished, forexample, using monoclonal antibodies that bind selectively to expressedCDs, antigens, receptors, and the like. Examples of tumor cell antigensinclude CD13 and CD33 present on myeloid cells; CD10 and CD19 present onB-cells; and CD2, CD5, and CD7 present on T-cells. One of skill in theart will recognize that numerous markers are available that identifyvarious known cell markers. Moreover, additional markers are continuallybeing discovered. Any such markers, whether known now or discovered inthe future, that are useful in labeling cells may be exploited inpracticing the invention.

Since few, if any markers are absolutely specific to only a single typeof cell, it may be desirable to label at least two markers, each with adifferent label, for each chosen cell type. Detection of multiple labelsfor each chosen cell type should help to ensure that the chemotaxis andchemoinvasion analysis is limited only to the cells of interest.

The present invention further provides a test device comprising: supportmeans; means mounted to the support means for defining a discretechamber with the support means by being placed in fluid-tight, conformalcontact with the support means. The discrete chamber includes a firstwell region including at least one first well; a second well regionincluding at least one second well, the second well region further beinghorizontally offset with respect to the first well region in a testorientation of the device; and a channel region including at least onechannel connecting the first well region and the second well region withone another. An example of the support means comprises the supportmember 16 shown in FIGS. 1A, 1B, 12 and 13, while an example of themeans mounted to the support means comprises the top member 11 shown inFIGS. 1A-11, 13 and 14. Other such means would be well known by personsskilled in the art.

From the foregoing, it will be observed that numerous modifications andvariations can be effected without departing from the true spirit andscope of the novel concept of the present invention. For example,different embodiments of a device of the present invention may becombined. Embodiments of the present invention further contemplatedifferent types of assays, for example, an assay wherein the test agentcomprises a buffer solution instead of a chemotactic agent. In such anassay, cell migration through channel region 15 a in observed in theabsence of a chemotactic gradient.

It will be appreciated that the present disclosure is intended to setforth the exemplifications of the invention, and the exemplificationsset forth are not intended to limit the invention to the specificembodiments illustrated. The disclosure is intended to cover by theappended claims all such modifications as fall within the spirit andscope of the claims.

EXAMPLES Example 1 Procedure for Fabrication of Chemoinvasion Device

A silicon wafer (6 inches) is spin coated with photoresist (SU8-50) at200 rpm for 45 seconds. After baking the wafer on a hot plate at 115° C.for 10 minutes, the wafer is allowed to cool to room temperature. A maskaligner (EVG620) is used to expose the photoresist film through aphotomask. Exposure of 45 seconds is followed by another hard bake at115° C. for 10 minutes. The silicon wafer is allowed to cool to roomtemperature for over 30 minutes. The uncrosslinked photoresist isremoved using propylene glycol methyl ether acetate (PGMEA). The waferis dried under a stream of nitrogen, and the patterned photoresist isready for subsequent processing.

In one embodiment, the patterned photoresist is spin-coated with anotherlayer of SU8-100 at 1500 rpm for 45 seconds. A mask aligner is used toselectively expose macrofeatures (i.e. wells) of the top member but notexpose channel regions connecting the wells and other areas of the topmember. After post exposure processing and photoresist removal, themaster contains multiple layered features. This step may be repeated tointroduce macro-features on the master, which have the height ofapproximately 3 mm.

When a PDMS prepolymer is cast against the master, it faithfullyreplicates the features in the master. When casting, PDMS is added in anamount slightly lower than the height of the macrofeatures. After curingthe PDMS for four hours at 65 degrees C., the PDMS is peeled off thesilicon master and thoroughly cleaned with soap and water and rinsedwith 100% ethanol. A glass support member is also cleaned and rinsedwith ethanol. The PDMS membrane and glass support member are plasmaoxidized for 1 minute with the sides that would be bonded togetherfacing upward. The PDMS membrane is then placed onto the glass supportmember and pressure is applied to remove any air bubbles that may haveformed between the PDMS membrane and the glass support member. Theassembled device is then cooled to 4° C. Within 15 minutes of the plasmaoxidation of the PDMS membrane and the glass support member, 20microliters (μl) of Matrigel (any other hydrogel may be used) is pouredinto the first well and allowed to flow into the capillaries. The deviceis placed at room temperature for 15 minutes to set the Matrigel. Excessgel is then removed from the wells of the top member using a vacuum anda Pasteur pipette.

Example 2 Cell Chemoinvasion Assay

Placement of Cells and Test Agent in Chamber

The first and second wells of a chamber of a top member are filled withphosphate buffered saline solution, PBS. The bottom of the second wellmay be treated with fibronectin (1 mg/ml) or other extracellular matrixprotein for 30 minutes, followed by washing twice with PBS. Afteraspirating PBS, astrocytoma cells (U87-MG) are plated in 50 μl offreshly warmed medium in the second well (25,000 cells per well of a24-well plate, in volume of 50 ul of solution per well). The cellsdeposit through the second well of the chamber, and attach to the bottomof the second well.

Cells are left to attach and spread in the second well overnight in a37° C. incubator. At the start of the experiment, the cell medium isexchanged for fresh serum-free medium. 10 μg of bFGF (basic fibroblastgrowth factor) per ml of medium is added to the first well of eachchamber.

Image Acquisition and Data Analysis

Digital Images are taken on a Zeiss inverted microscope using AXIOCAM™.Data was analyzed on AXIOVISION™ software. Time-lapsed images are takenevery day at the same time for four days.

Example 3 Cell Chemoinvasion Inhibition Assay Using Solution Gradient

Placement of Cells and Test Agent in Chambers

With respect to three chambers, the wells of each chamber of a topmember are filled with PBS. The bottom of the second wells may betreated with fibronectin (1 mg/ml) or other extracellular matrix proteinfor 30 minutes, followed by washing twice with PBS. After aspiratingPBS, U87-MG cells are plated in 50 μl of freshly warmed medium in thesecond wells (10,000 cells per well of a 24-well plate, in volume of 50μl of medium per well). The cells deposit through the second wells ofeach chamber, and adhere to the bottom of the second wells.

Cells are left to attach and spread in the second wells overnight in a37° C. incubator. At the start of the experiment, the cell medium isexchanged for fresh serum-free medium or 1% serum. 1 μg of bFGF (basicfibroblast growth factor) per ml of medium is added to the first wellsof the chamber. A solution gradient is allowed to form for one hour.

With respect to the three different chambers, 100 μM of LY294002 areplaced in the second well of chamber #1, 10 μM LY294002 of are placed inthe second well of chamber #2, and 1.0 μM of LY294002 are placed in thesecond well of chamber #3.

Image Acquisition and Data Analysis

Digital Images are taken on a Zeiss inverted microscope using AXIOCAM™.Data was analyzed on AXIOVISION™ software. Time-lapsed images are takenevery day at the same time for four days.

Example 4

Immobilization of Biomolecules on Support Member

After assembling the device as described above, the channel regions arefilled with ethanolic solution containing (CH₃CH₂O)₃Si (CH₂)₃NH₂. After20 minutes at room temperature, the channel regions are washed off usingethanol. The device is incubated at 105° C. for one hour to crosslinkthe siloxane monolayer formed on the support member. The device iswashed with ethanol to remove residues. The channel regions are filledwith a solution of diisocyanate, either hexamethylene diisocyanate ortolyl diisocyanate (1% in acetonitrile or N-methyl pyrrolidinone). Thediisocyanate is allowed to react for two hours with the terminal aminogroups of the siloxane monolayer formed on the support member. Thediisocyanate is washed off. The channel regions are filled with 1 mg/mlsolution of heparan sulfate or other sulfated carbohydrates (forexample, di-acetylated form of heparin, heparin fragments, lectinscontaining sulfated sugars, etc.) The heparan sulfate is allowed toreact with the support member to form immobilized species. The heparansulfate solution and other reagents are washed off. A chemokine solution(any chemokine from CC, CXC, CX3C, or XC families may be used) isintroduced into the channel region. By electrostatic interaction,chemokines that have higher pI (˜9-10) adsorb onto the negativelycharged sulfated support member.

Example 5 Chemotaxis Inhibition Assay Using Surface Gradient

Two wells are filled with 50 μl of PBS, and hydrostatic pressure isallowed to equalize. 5 μl of anti-hisx6 antibody are added to the firstwell and 5 μl of buffer are added to the second well to equalizehydrostatic pressure. By diffusion, the antibody concentration forms agradient from the first well to the second well. After 2 hours at roomtemperature, the two wells are washed off by adding 50 μl of buffer tothe second well and removing 50 μl from the first well. Byphysisorption, the solution gradient is transferred onto a surfacethereby forming a surface gradient. A solution of IL-8 (recombinanthuman IL-8 with a HISx6 fusion tag, R+D systems, catalog No. 968-IL) atconcentration of 25 μg/ml is added to the channel regions. The solutionis allowed to incubate for 30 minutes at room temperature. Excess IL-8chemokine is washed off and the surface is decorated with bound IL-8.Neutrophils(freshly isolated from a healthy donor) are added to thesecond well. Typically 20,000-100,000 cells are added in volume rangingfrom 10-550 μl. Neutrophils are allowed to adhere to the support memberand allowed to migrate towards the higher concentration of IL-8.Inhibition of migration is achieved by adding polyclonal antibodyagainst IL-8.

1. A device for monitoring haptotaxis including a housing comprising: asupport member; a top member, the top member mounted to the supportmember wherein the support member and the top member are configured suchthat they together define at least one discrete chamber, the at leastone discrete chamber including: a first well region including at leastone first well, the at least one first well configured to receive a testagent therein, the at least one first well including a first pluralityof biomolecules immobilized therein; a second well region including atleast one second well, the at least one second well configured toreceive a sample comprising cells therein and further being horizontallyoffset with respect to the first well region in a test orientation ofthe device; and a channel region including only one channel connectingthe first well region and the second well region, the one channelincluding a second plurality of biomolecules immobilized therein, theconcentration of the first plurality of biomolecules in the first wellbeing greater than the concentration of the second plurality ofbiomolecules in the one channel, and the concentration decreasing fromthe first well region towards the second well region forming a surfaceconcentration gradient of the first and second plurality of immobilizedbiomolecules, wherein the first plurality of biomolecules and the secondplurality of biomolecules are of the same type.
 2. The device of claim1, wherein the plurality of biomolecules are chemokines, cytokines, andsmall molecules.
 3. The device of claim 1, wherein the one channelcontains a gel matrix.
 4. The device of claim 1, wherein the top memberis in reversible, fluid tight conformal contact with the support member.5. The device of claim 1, wherein the top member is made of anelastomeric material.
 6. The device of claim 5, wherein the top memberis made of PDMS.
 7. The device of claim 1, wherein the discrete chamberis a plurality of discrete chambers.
 8. The device of claim 7, whereinthe first well regions and the second well regions of the plurality ofdiscrete chambers are disposed relative to one another to match a pitchof a standard microtiter plate.
 9. The device of claim 7, wherein theplurality of discrete chambers are disposed relative to one another tomatch a pitch of a standard microtiter plate.
 10. The device of claim 1,wherein the one channel has at least one of: a length between about 3microns to about 18 mm; a width between about 3 microns to about 200microns; and a depth between about 3 microns to about 200 microns. 11.The device of claim 1, wherein the at least one channel has both a widthand a depth of about 3 microns to about 20 microns.
 12. The device ofclaim 3, wherein the at least one channel has both a width and a depthof about 20 microns to about 100 microns.
 13. The device of claim 3,wherein the at least one channel has both a width and a depth of about20 microns to about 200 microns.
 14. The device of claim 1, wherein theat least one channel has a length between about 100 microns to about 3mm, a width between about 3 microns to about 200 microns, and a depth ofabout 3 microns to about 20 microns.
 15. The device of claim 1, whereina width of at least one of the first well region and the second wellregion is smaller than a length of said at least one of the first wellregion and the second well region.
 16. The device of claim 1, whereinthe support member is a glass member having a thickness between about0.1 to about 2 mm.
 17. The device of claim 1, wherein, in a testorientation of the device, the support member defines at least onepredetermined upper surface region that is sloped with respect to ahorizontal plane.
 18. The device of claim 17, wherein the at least onepredetermined upper surface region of the support member includes abottom surface of one of the first well region and the second wellregion, the bottom surface being sloped downward in a direction towardthe at least one channel.
 19. The device of claim 17, where the at leastone predetermined upper surface region of the support member includesbottom surfaces of respective ones of the first well region and thesecond well region, each of the bottom surfaces being sloped downward ina direction toward the at least one channel.
 20. The device of claim 17,wherein the at least one predetermined upper surface region is sloped soas to define an angle of less than 90 degrees with respect to thehorizontal plane.
 21. The device of claim 1, further comprising a rigidframe placed around an outer perimeter of the top member.
 22. A devicefor monitoring haptotaxis including a housing defining a discretechamber, the chamber having an opening facing vertically upward in atest orientation of the device and the chamber further comprising: afirst well region including at least one first well, the at least onefirst well configured to receive a test agent therein, the at least onefirst well including a first plurality of biomolecules immobilizedtherein; a second well region including at least one second well, thesecond well region further being horizontally offset with respect to thefirst well region in a test orientation of the device, the at least onesecond well configured to receive a sample comprising cells therein; anda channel region including only one channel connecting the first wellregion and the second well region, the one channel including a secondplurality of biomolecules immobilized therein, the concentration of thefirst plurality of biomolecules in the first well being greater than theconcentration of the second plurality of biomolecules in the onechannel, and the concentration decreasing from the first well regiontowards the second well region forming a surface concentration gradientof the first and second plurality of immobilized biomolecules, whereinthe first plurality of biomolecules and the second plurality ofbiomolecules are of the same type.
 23. A device for monitoringhaptotaxis comprising: support means; means mounted to the support meansfor defining a discrete chamber with the support means, the discretechamber including: a first well region including at least one firstwell, the at least one first well configured to receive a test agenttherein, the at least one first well including a first plurality ofbiomolecules immobilized therein; a second well region including atleast one second well, the second well region further being horizontallyoffset with respect to the first well region in a test orientation ofthe device, the at least one second well configured to receive a samplecomprising cells; and a channel region including only one channelconnecting the first well region and the second well region with oneanother, the one channel and the first well region including a pluralityof biomolecules immobilized therein, the concentration of the pluralityof biomolecules decreasing from the first well region towards the secondwell region forming a surface concentration gradient of the plurality ofimmobilized biomolecules. a channel region including only one channelconnecting the first well region and the second well region, the onechannel including a second plurality of biomolecules immobilizedtherein, the concentration of the first plurality of biomolecules in thefirst well being greater than the concentration of the second pluralityof biomolecules in the one channel, and the concentration decreasingfrom the first well region towards the second well region forming asurface concentration gradient of the first and second plurality ofimmobilized biomolecules, wherein the first plurality of biomoleculesand the second plurality of biomolecules are of the same type.
 24. Thedevice of claim 23, wherein: the support means comprises a supportmember; and the means for defining comprises a top member.
 25. A devicefor monitoring haptotaxis comprising: a support member; a top membermounted to the support member by forming a substantially instantaneousseal with the support member, wherein the support member and the topmember are configured such that they together define a discrete chamber,the discrete chamber including: a first well region including at leastone first well, the at least one first well configured to receive a testagent therein, the at least one first well including a first pluralityof biomolecules immobilized therein; a second well region including atleast one second well, the second well region configured to receive asample comprising cells therein and further being horizontally offsetwith respect to the first well region in a test orientation of thedevice; and a channel region including only one channel connecting thefirst well region and the second well region, the one channel includinga second plurality of biomolecules immobilized therein, theconcentration of the first plurality of biomolecules in the first wellbeing greater than the concentration of the second plurality ofbiomolecules in the one channel, and the concentration decreasing fromthe first well region towards the second well region forming a surfaceconcentration gradient of the first and second plurality of immobilizedbiomolecules, wherein the first plurality of biomolecules and the secondplurality of biomolecules are of the same type.
 26. A device formonitoring haptotaxis including a housing defining a chamber, thechamber comprising: a first well region including at least one firstwell, the first well configured to receive a test agent therein, the atleast one first well including a first plurality of biomoleculesimmobilized therein; a second well region including at least one secondwell, the second well region configured to receive a sample comprisingcells therein and further being horizontally offset with respect to thefirst well region in a test orientation of the device; and a channelregion including only one channel connecting the first well region andthe second well region, the only channel including a second plurality ofbiomolecules immobilized therein, the concentration of the firstplurality of biomolecules in the first well being greater than theconcentration of the second plurality of biomolecules in the onechannel, and the concentration decreasing from the first well regiontowards the second well region forming a surface concentration gradientof the first and second plurality of immobilized biomolecules, whereinthe first plurality of biomolecules and the second plurality ofbiomolecules are of the same type.
 27. A device for monitoringhaptotaxis including a housing comprising: a support member; a topmember, the top member mounted to the support member wherein the supportmember and the top member are configured such that they together defineat least one discrete chamber, the at least one discrete chamberincluding: a first well region including at least one first well, the atleast one first well configured to receive a test agent therein andfurther including a plurality of biomolecules immobilized therein; asecond well region including at least one second well, the at least onesecond well configured to receive a sample comprising cells therein andfurther being horizontally offset with respect to the first well regionin a test orientation of the device; and a channel region comprising aplurality of channels connecting the first well region and the secondwell region with one another, the plurality of channels and the firstwell region including a plurality of biomolecules decreasing from thefirst well region towards the second well region forming a surfaceconcentration gradient of immobilized biomolecules, wherein none of theplurality of channels diverge from each other or converge with eachother.
 28. The device of claim 27, wherein the plurality of channelshave an identical length with respect to one another.
 29. The device ofclaim 27, wherein the length of each one of the plurality of channelssuccessively increases in a direction of the at least one second wellsuch that respective channel inlets at one of the first well region andthe second well region are aligned.
 30. The device of claim 27, whereinthe at least one first well is a plurality of first wells, each of theplurality of first wells connected to another one of the plurality offirst wells and each of the plurality of first wells including theplurality of immobilized biomolecules therein; the at least one secondwell is a plurality of second wells, each of the plurality of secondwells corresponding to a respective one of the plurality of first wellsand each of the plurality of second wells configured to receive asamples comprising cells; and the plurality of channels connects arespective one of the plurality of first wells with a corresponding oneof the plurality of second wells and each of the plurality of channelsincludes the plurality of immobilized biomolecules therein, theconcentration of the plurality of biomolecules decreasing from the firstwell region towards the second well region forming a surfaceconcentration gradient of the plurality of immobilized biomolecules. 31.The device of claim 30, wherein at least one of the plurality ofchannels defines a plurality of subchannels therein each of theplurality of channels including immobilized biomolecules therein, theconcentration of biomolecules decreasing along the longitudinal axis ofthe device from the first well region towards the second well regionforming a surface concentration gradient of immobilized biomolecules.32. The device of claim 27, wherein at least two of the plurality ofchannels have at least one of different shapes and different dimensionswith respect to one another.
 33. The device of claim 27, wherein thelength of each of the plurality of channels successively increases in adirection of the second well.
 34. The device of claim 27, wherein thewidth of each of the plurality of channels successively increases in adirection of the at least one second well.
 35. The device of claim 27,wherein the depth of each of the plurality of channels successivelyincreases in a direction of the at least one second well.
 36. A devicefor monitoring haptotaxis including a housing comprising: a supportmember; a top member, the top member being in reversible, fluid-tightconformal contact with the support member wherein the support member andthe top member are configured such that they together define at leastone discrete chamber, the at least one discrete chamber including: afirst well region including at least one first well, the at least onefirst well configured to receive a test agent therein, the at least onefirst well including a first plurality of biomolecules immobilizedtherein; a second well region including at least one second well, the atleast one second well configured to receive a sample comprising cellstherein and further being horizontally offset with respect to the firstwell region in a test orientation of the device; and a channel regionincluding only one channel connecting the first well region and thesecond well region, the one channel including a second plurality ofbiomolecules immobilized therein, the concentration of the firstplurality of biomolecules in the first well being greater than theconcentration of the second plurality of biomolecules in the onechannel, and the concentration decreasing from the first well regiontowards the second well region forming a surface concentration gradientof the first and second plurality of immobilized biomolecules, whereinthe first plurality of biomolecules and the second plurality ofbiomolecules are of the same type.